The majority of phosphorous (P) in farm animal feed grains is present as a mixed salt of myo-inositol hexakisphosphate, shown below, and more commonly referred to as phytic acid (“myo-IP6”).

Because grain-consuming animals such as swine and poultry do not produce the enzyme phytase, myo-IP6 is largely unavailable as a phosphorus-containing nutrient source and is thus excreted in high concentrations in the waste of these animals. Such waste is, however, applied to croplands as a means of enhancing soil fertility, though little information exists regarding the process of how myo-IP6 is transformed into a crop-available nutrient. At least in part, this lack of information respecting the fate of myo-IP6 in soil and water-sediment environments is attributable to the absence of artificial substrates that can be used for the convenient measurement of phytase activity. Still, recent experimental evidence suggests that bacterial phytase plays an important role in the (bio)chemical transformation.
Phytases catalyze the sequential hydrolysis of myo-IP6, forming orthophosphate (ortho-P) and a series of partially dephosphorylated phosphoric esters of myo-inositol. In some cases, hydrolysis may go to completion yielding the parent compound myo-inositol. Based on biochemical properties of the amino acid sequence alignment, others have characterized phytases into two major classes, the histidine acid phytases (comprising the PhyA, PhyB, and PhyC groups), to which most of the bacterial and fungal phytases belong, and the alkaline phytases (PhyD). The phytate-degrading enzyme, 3-phytase (myo-inositol hexakisphosphate 3-phosphohydrolase, EC 3.1.3.8; PhyA and PhyB groups) hydrolyzes myo-IP6 preferentially at the C-3 position, while 6-phytase (myo-inositol hexakisphsophate 6-phosphohydrolase, EC 3.1.3.26; PhyC) hydrolyzes myo-IP6 preferentially at the C-6 position.
Conventional phytase assays conducted on bacterial cell wall-free lysate or whole cell lysate routinely call for the addition of myo-IP6 to buffered cell lysate and subsequent measurement of the released ortho-P by colorimetric analysis. However, experiments by the inventor hereof demonstrates substantial ortho-P release from bacterial cell lysate resulting from “cell free” phosphate-mediated hydrolysis of cell-associated phosphate compounds, including accumulated intracellular polyphosphate. Notably, bacteria, archaea and fungi are all capable of producing polyphosphate. Thus, it would seem that the conventional ortho-P release assay is not a specific measure of phytase activity in lysed cell preparations, and its use under such conditions may result in an exaggerated estimate of phytase activity or, possibly, even a false positive test result.
In light of the foregoing, it would be desirable to provide a specific and sensitive quantitative enzyme assay capable of measuring phytase activity in cell culture filtrates, cell-lysate preparations, soils, etc.